Gaining Insights into Inherited Bleeding Disorders of Complex Etiology in Pediatric Patients: Whole-Exome Sequencing as First-Line Investigation Tool.

INTRODUCTION: Investigation of the molecular basis of inherited bleeding disorders (IBD) is mostly performed with gene panel sequencing. However, the continuous discovery of new related genes underlies the limitation of this approach. This study aimed to identify genetic variants responsible for IBD in pediatric patients using whole-exome sequencing (WES), and to provide a detailed description and reclassification of candidate variants. MATERIAL AND METHODS: WES was performed for 18 pediatric patients, and variants were filtered using a first-line list of 290 genes. Variant prioritization was discussed in a multidisciplinary team based on genotype-phenotype correlation, and segregation studies were performed with available family members. RESULTS: The study identified 22 candidate variants in 17 out of 18 patients (94%). Eleven patients had complete genotype-phenotype correlation, resulting in a diagnostic yield of 61%, 5 (28%) were classified as partially solved, and 2 (11%) remained unsolved. Variants were identified in platelet (ACTN1, ANKRD26, CYCS, GATA1, GFI1B, ITGA2, NBEAL2, RUNX1, SRC, TUBB1), bleeding (APOLD1), and coagulation (F7, F8, F11, VWF) genes. Notably, 9 out of 22 (41%) variants were previously unreported. Variant pathogenicity was assessed according to the American College of Medical Genetics and Genomics guidelines and reclassification of three variants based on family segregation evidence, resulting in the identification of 10 pathogenic or likely pathogenic variants, 6 variants of uncertain significance, and 6 benign or likely benign variants. CONCLUSION: This study demonstrated the high potential of WES in identifying rare molecular defects causing IBD in pediatric patients, improving their management, prognosis, and treatment, particularly for patients at risk of malignancy and/or bleeding due to invasive procedures.

Key Genes of the Immune System and Predisposition to Acquired Hemophilia A: Evidence from a Spanish Cohort of 49 Patients Using Next-Generation Sequencing.

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by the presence of autoantibodies against factor VIII (FVIII). As with other autoimmune diseases, its etiology is complex and its genetic basis is unknown. The aim of this study was to identify the immunogenetic background that predisposes individuals to AHA. HLA and KIR gene clusters, as well as KLRK1, were sequenced using next-generation sequencing in 49 AHA patients. Associations between candidate genes involved in innate and adaptive immune responses and AHA were addressed by comparing the alleles, genotypes, haplotypes, and gene frequencies in the AHA cohort with those in the donors' samples or Spanish population cohort. Two genes of the HLA cluster, as well as rs1049174 in KLRK1, which tags the natural killer (NK) cytotoxic activity haplotype, were found to be linked to AHA. Specifically, A*03:01 (p = 0.024; odds ratio (OR) = 0.26[0.06-0.85]) and DRB1*13:03 (p = 6.8 × 103, OR = 7.56[1.64-51.40]), as well as rs1049174 (p = 0.012), were significantly associated with AHA. In addition, two AHA patients were found to carry one copy each of the low-frequency allele DQB1*03:09 (nallele = 2, 2.04%), which was completely absent in the donors. To the best of our knowledge, this is the first time that the involvement of these specific alleles in the predisposition to AHA has been proposed. Further molecular and functional studies will be needed to unravel their specific contributions. We believe our findings expand the current knowledge on the genetic factors involved in susceptibility to AHA, which will contribute to improving the diagnosis and prognosis of AHA patients.

Impact of genetic structural variants in factor XI deficiency: identification, accurate characterization, and inferred mechanism by long-read sequencing.

BACKGROUND: Congenital factor XI (FXI) deficiency is a probably underestimated coagulopathy that confers antithrombotic protection. Characterization of genetic defects in F11 is mainly focused on the identification of single-nucleotide variants and small insertion/deletions because they represent up to 99% of the alterations accounting for factor deficiency, with only 3 gross gene defects of structural variants (SVs) having been described. OBJECTIVES: To identify and characterize the SVs affecting F11. METHODS: The study was performed in 93 unrelated subjects with FXI deficiency recruited in Spanish hospitals over a period of 25 years (1997-2022). F11 was analyzed by next-generation sequencing, multiplex ligand probe amplification, and long-read sequencing. RESULTS: Our study identified 30 different genetic variants. Interestingly, we found 3 SVs, all heterozygous: a complex duplication affecting exons 8 and 9, a tandem duplication of exon 14, and a large deletion affecting the whole gene. Nucleotide resolution obtained by long-read sequencing revealed Alu repetitive elements involved in all breakpoints. The large deletion was probably generated de novo in the paternal allele during gametogenesis, and despite affecting 30 additional genes, no syndromic features were described. CONCLUSION: SVs may account for a high proportion of F11 genetic defects implicated in the molecular pathology of congenital FXI deficiency. These SVs, likely caused by a nonallelic homologous recombination involving repetitive elements, are heterogeneous in both type and length and may be de novo. These data support the inclusion of methods to detect SVs in this disorder, with long-read-based methods being the most appropriate because they detect all SVs and achieve adequate nucleotide resolution.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project.Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p = 3.7 × 10-6), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10-10) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood (p = 6.14 × 10-9). Additionally, ORM1 expression correlated with levels of cfDNA (p = 4.38 × 10-4). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association (ρ g = 0.43, p = 0.089).All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Molecular study of a large cohort of 109 haemophilia patients from Cuba using a gene panel with next generation sequencing-based technology.

INTRODUCTION: In several countries, molecular diagnosis of haemophilia A (HA) and B (HB) is hampered by a lack of resources for DNA analysis. The advent of next-generation sequencing (NGS) has enabled gene analysis at a reasonable cost. AIM: Describe a collaboration between Cuban and Spanish researchers to identify candidate variants and investigate the molecular epidemiology of 106 Cuban haemophilia patients using NGS. PATIENTS/METHODS: The molecular analysis protocol included well-established LR-PCR procedures to detect F8 inversions, NGS with a 30-gene panel to sequence F8 and F9, and multiplex ligation-dependent probe amplification to identify large structural variants. RESULTS: One-hundred and thirty-one candidate variants were identified along F8, F9, and VWF; 72 were unique and 28 (39%) had not been previously recorded. Putative variants were identified in 105/106 patients. Molecular characterization enabled confirmation and reclassification of: 90 HA (85%), 15 HB (14%), and one type 2N VWD (1%). Null variants leading to non-production of FVIII or FIX were common in severe HA (64%), moderate HA (74%), and severe HB (60%), whereas missense variants were frequent in mild HA (57%) and moderate or mild HB (83%). Additional variants in VWF were identified in 16 patients. CONCLUSION: This is the first description of the molecular epidemiology of HA and HB in Cuba. Variants identified in index cases will be of value for local implementation of familial studies and prenatal diagnosis using the molecular approaches available in Cuba. The results of this protocolled genetic study improved the accuracy of the clinical diagnosis and will facilitate management of these patients.

Next generation sequencing to dissect the genetic architecture of KNG1 and F11 loci using factor XI levels as an intermediate phenotype of thrombosis.

Venous thromboembolism is a complex disease with a high heritability. There are significant associations among Factor XI (FXI) levels and SNPs in the KNG1 and F11 loci. Our aim was to identify the genetic variation of KNG1 and F11 that might account for the variability of FXI levels. The KNG1 and F11 loci were sequenced completely in 110 unrelated individuals from the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project using Next Generation Sequencing on an Illumina MiSeq. The GAIT-2 Project is a study of 935 individuals in 35 extended Spanish families selected through a proband with idiopathic thrombophilia. Among the 110 individuals, a subset of 40 individuals was chosen as a discovery sample for identifying variants. A total of 762 genetic variants were detected. Several significant associations were established among common variants and low-frequency variants sets in KNG1 and F11 with FXI levels using the PLINK and SKAT packages. Among these associations, those of rs710446 and five low-frequency variant sets in KNG1 with FXI level variation were significant after multiple testing correction and permutation. Also, two putative pathogenic mutations related to high and low FXI levels were identified by data filtering and in silico predictions. This study of KNG1 and F11 loci should help to understand the connection between genotypic variation and variation in FXI levels. The functional genetic variants should be useful as markers of thromboembolic risk.

The Central Role of KNG1 Gene as a Genetic Determinant of Coagulation Pathway-Related Traits: Exploring Metaphenotypes.

Traditional genetic studies of single traits may be unable to detect the pleiotropic effects involved in complex diseases. To detect the correlation that exists between several phenotypes involved in the same biological process, we introduce an original methodology to analyze sets of correlated phenotypes involved in the coagulation cascade in genome-wide association studies. The methodology consists of a two-stage process. First, we define new phenotypic meta-variables (linear combinations of the original phenotypes), named metaphenotypes, by applying Independent Component Analysis for the multivariate analysis of correlated phenotypes (i.e. the levels of coagulation pathway-related proteins). The resulting metaphenotypes integrate the information regarding the underlying biological process (i.e. thrombus/clot formation). Secondly, we take advantage of a family based Genome Wide Association Study to identify genetic elements influencing these metaphenotypes and consequently thrombosis risk. Our study utilized data from the GAIT Project (Genetic Analysis of Idiopathic Thrombophilia). We obtained 15 metaphenotypes, which showed significant heritabilities, ranging from 0.2 to 0.7. These results indicate the importance of genetic factors in the variability of these traits. We found 4 metaphenotypes that showed significant associations with SNPs. The most relevant were those mapped in a region near the HRG, FETUB and KNG1 genes. Our results are provocative since they show that the KNG1 locus plays a central role as a genetic determinant of the entire coagulation pathway and thrombus/clot formation. Integrating data from multiple correlated measurements through metaphenotypes is a promising approach to elucidate the hidden genetic mechanisms underlying complex diseases.

The Unravelling of the Genetic Architecture of Plasminogen Deficiency and its Relation to Thrombotic Disease.

Although plasminogen is a key protein in fibrinolysis and several mutations in the plasminogen gene (PLG) have been identified that result in plasminogen deficiency, there are conflicting reports to associate it with the risk of thrombosis. Our aim was to unravel the genetic architecture of PLG in families with plasminogen deficiency and its relationship with spontaneous thrombotic events in these families. A total of 13 individuals from 4 families were recruited. Their genetic risk profile of thromboembolism was characterized using the Thrombo inCode kit. Only one family presented genetic risk of thromboembolism (homozygous carrier of F12 rs1801020 and F13A1 rs5985). The whole PLG was tested using Next Generation Sequencing (NGS) and 5 putative pathogenic mutations were found (after in silico predictions) and associated with plasminogen deficiency. Although we can not find genetic risk factors of thrombosis in 3 of 4 families, even the mutations associated with plasminogen deficiency do not cosegregated with thrombosis, we can not exclude plasminogen deficiency as a susceptibility risk factor for thrombosis, since thrombosis is a multifactorial and complex disease where unknown genetic risk factors, in addition to plasminogen deficiency, within these families may explain the thrombotic tendency.

Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project.

BACKGROUND: Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE. OBJECTIVES: To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE. PATIENTS/METHODS: Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes. RESULTS: The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed. CONCLUSIONS: The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results.

A Rao-Blackwellized particle filter for joint parameter estimation and biomass tracking in a stochastic predator-prey system.

Functional response estimation and population tracking in predator-prey systems are critical problems in ecology. In this paper we consider a stochastic predator-prey system with a Lotka-Volterra functional response and propose a particle filtering method for: (a) estimating the behavioral parameter representing the rate of effective search per predator in the functional response and (b) forecasting the population biomass using field data. In particular, the proposed technique combines a sequential Monte Carlo sampling scheme for tracking the time-varying biomass with the analytical integration of the unknown behavioral parameter. In order to assess the performance of the method, we show results for both synthetic and observed data collected in an acarine predator-prey system, namely the pest mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis.

Linkage and association analyses using families identified a locus affecting an osteoporosis-related trait.

Osteoporosis is a common disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in an increase in bone fragility and in susceptibility to fractures. The genetic basis of osteoporosis is complex and involves multiple genes and environmental factors. Here we introduce a family-based study of the genetics of osteoporosis - the Genetic Analysis of Osteoporosis (GAO) Project - to discover genetic variants affecting osteoporosis-related phenotypes. The GAO Project involved 11 extended families from Barcelona, Spain selected through a proband with osteoporosis (N=367). We performed spine, femur and whole body densitometry for all participants and also analyzed strength and geometrical properties of the hip. Our study focused on 23 densitometric phenotypes that we considered of high clinical relevance and four definitions of low bone mass and fracture status. Pedigree validation was carried out through microsatellite genotyping. The same microsatellites were used to interrogate our data (i) for the replication of previous linkage signals and (ii) for the potential discovery of new linkage signals. The linkage analysis identified one region marked by microsatellite D17S787 showing a strong and significant signal of linkage with femoral shaft cross-sectional moment of inertia (CSMI; LOD=3.18; p=6.5×10(-5)). The chromosomal location marked by microsatellite D17S787 includes several genes, among which two are of particular interest: COL1A1 and SOST, coding for collagen alpha-1 (I) chain and sclerostin, respectively. Follow-up association analysis resulted in only one significant result for rs4792909 from the SOST genomic region (p=0.00248). As a result, we provide strong and significant evidence from both linkage and association analyses that the SOST gene may affect the strength of the femoral shaft. Future investigations should study the relationship between bone mass formation and strength properties of the bones.